Difference between revisions of "Part:BBa K608351:Experience"

Revision as of 04:15, 16 October 2016

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User Reviews

team:Tokyo Tech 2016

Registered sequence PSB6A1 has lost 272 bases downstream from its recognition site of PstI. When lasR (BBa_C0179) was inserted into pSB6A1, ligation was confirmed cutting by SalI. At that time, each of lasR and pSB6A1 is cut at one site, and 800 bases are produced. From the result of an electrophoresis, it has bases from 500 to 600 (instead of 800 bases.) The result of sequencing shows that the recognition sequence of PstI and SalI are adjacent.

Fig. 1. Agarose gel electrophoresis of lasR (pSB6A1) cut with SalI. Lane 1 shows that approximately 300 bases are missing in lasR or pSB6A1.

The expected of pSB6A1 sequence would be this.

Applications of BBa_K608351

We managed to perform a last-minute, "blind" -ie not knowing if the sequence of the insert was correct-, and preliminary OD-measurement testing of the part in conjuction with (BBa_K608352), as part of our temperature sensitive phage lysis cassette.

We later verified the part sequence. Please refer to our Project Results Page (http://2011.igem.org/Team:Freiburg/Results#Lysis_cassette) for more Info.

The OD values of the measurement are shown in the chart (please do bear in mind that this was a measurement done under deadline-stress, without the possibility to plan and perform something more concrete before the Wiki-Freeze)

LB with Cm (Chloramphenicol) was used as a blank before every (hourly) measurement

Nr1: Insert is only this part ((BBa_K608351))
Nr2: Insert is only (BBa_K608352)
Nr3: Both parts were correctly assembled (1:(BBa_K608351) + 2:(BBa_K608352))

Therefore (BBa_K608351) seems to be functioning, though not tightly. More tests are planned if time allows (ie wiki-unfreeze ;) ) One sure thing is that the correct testing would be done with GFP or RFP instead of (BBa_K608352), and also at different temperatures (probably a 25°C to 42°C gradient).

User Reviews

UNIQ5568f1cc47d444bd-partinfo-00000000-QINU UNIQ5568f1cc47d444bd-partinfo-00000001-QINU

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leony16111710

Activation Temperature

This image shows multiple tests of the Temperature Sensitive Promoter (pTS) with a GFP generator (GFP gen) attached downstream. The goal was to determine what temperature activates the Temperature Sensitive Promoter. The pictures on the left show the pTS+GFP gen construct grown at different temperatures including 30, 32, 35, 36, 37, 42 Celsius under white light conditions. The pictures on the right show the same plates under blue light, where only the 37 and 42 Celsius glowed. This indicates that GFP was only found at or above 37 Celsius.

Activation of Temperature Sensitive Promoter at Various Temperatures

At around 3hrs the green fluorescence of the pTS + GFP gen plate becomes visible (diverges from that of the negative control towards that of the positive control).

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 ===User Reviews===
+team:<b>Tokyo Tech 2016</b>
 Registered sequence PSB6A1 has lost 272 bases downstream from its recognition site of PstI. When lasR (BBa_C0179) was inserted into pSB6A1, ligation was confirmed cutting by SalI. At that time, each of lasR and pSB6A1 is cut at one site, and 800 bases are produced. From the result of an electrophoresis, it has bases from 500 to 600 (instead of 800 bases.) The result of sequencing shows that the recognition sequence of PstI and SalI are adjacent.